AU2006224660A1 - Mixture of at least 6 species of lactic acid bacteria and/or bifidobacteria in the manufacture of sourdough - Google Patents

Description

WO 2006/097415 PCT/EP2006/060505 1 MIXTURE OF AT LEAST 6 SPECIES OF LACTIC ACID BACTERIA AND/OR BIFIDOBACTERIA IN THE MANUFACTURE OF SOURDOUGH The present invention relates to the manufacture of baked goods and, more in general, starchy food. It provides baked goods and other food which are more digestible, they are gluten-free or have a reduced and markedly hydrolyzed gluten content and are particularly suitable for subjects affected by celiac disease. Background of the invention Cereals are important components of the daily diet. Nevertheless wheat flour gluten, and in particular the gliadin fraction, are responsi ble for human intolerance. The celiac disease, also known as Celiac Sprue (CS) or gluten-sensitive enteropathy, is one of the diffuse food intolerance, occurring in 1 out of every 130 to 300 persons of the Euro pean and U.S. populations. In South America, North Africa and Asia, is generally underestimated (Fasano and Catassi; 2001, Gastroenterol ogy, 120.636-651). The epidemiological distribution of CS is efficiently conceptualized by the iceberg model introduced by Logan in 1992 (Logan; 1992, Dyn. Nutr. Res. 2.14-24) where the prevalence of the dis ease is influenced by the frequency of the predisposing genotypes in the population. Total lifelong avoidance of gluten ingestion remains the cornerstone of CS treatment. The International Food Authority has now redefined the term gluten-free as zero tolerance for gluten, while the Codex Alimentarius permits a concentration of 200 ppm of gluten per food. Efforts to reduce the human intolerance to cereals are of a great medical, nutritional and economic interest. This is particularly true in the current context where the bakery industries are using very fast technological processes which may have an influence in the ex panding epidemiology of CS. Therefore, the need of more digestible and tolerated bread and baked products is really felt.

WO 2006/097415 PCT/EP2006/060505 2 CS is an autoimmune disease of the small intestinal mucosa in geneti cally susceptible persons. Upon ingestion of gluten, these patients suf fer from a self-perpetuating mucosal inflammation characterized by progressive loss of absorptive villi and hyperplasia of the crypts (Si lano and De Vincenzi, 1999; Nahrung, 43.175-184). During endolu minal proteolytic digestion, for instance gliadins of wheat release a family of Pro- and Glu-rich oligopeptides that are responsible for the T cell mediated immune response and/or, more in general, for the in flammatory state which characterizes the initial stage of CS (Silano and De Vincenzi; 1999). The literature reports the identification of the following oligopeptides: fragment 31-43 of the A-gliadin (Silano and De Vincenzi; 1999), fragment 62-75 of the a2-gliadin (Auricchio, S., et al.; 1996, Scand. J. Gastroenterol., 31:247-253; Picarelli, A., et al.; 1999, Scand. J. Gastroenterol., 34.1099-1102), the epitope 33-mer, which cor responds to the fragment 57-89 of the a2-gliadin (Shan, L., et al.; 2002. Science, 297.2275-2279), fragment 134-153 of y-gliadin (Aleanzi, M., et al.; 2001, Clin. Chem., 47. 2023-2028) and the fragment 57-68 of aX9 gliadin (Arentz-Hansen, et al.; 2000, J. Exp. Med., 191:603-612). Flours that are not tolerated by CS patients included wheat, rye, barley, ka mut, triticale and spelt. Some controversy is still debated for oat. Multidisciplinary research efforts are carried out in several fields to manage with CS. They concern the engineering of gluten free-grains (Fasano, A., et al.; 2003, Arch. Intern. Med., 163.286-292), search for the CS genes in humans (Fasano, A., et al.; 2003), use of some protec tive substances such as mannans and oligomers of N acetylglucosammine and the use of a bacterial prolyl-endopeptidase from Flavobacterium meningosepticum as an oral supplementary ther apy (Shan, et al.; 2002). More recently, two articles showed the extensive hydrolysis of the gli adin fractions by selected sourdough lactobacilli such as Lactobacillus alimentarius 15M, L. brevis 14G, L. sanfranciscensis 7A and L. hil gardii 51B (Di Cagno, et al.; 2002, Appl. Environ. Microbiol., 68:623 33) and, especially, the tolerance of 17 CS patients to breads which WO 2006/097415 PCT/EP2006/060505 3 contained 2 g of gluten, as determined by acute in vivo challenges based on the intestinal permeability (Di Cagno, et al.; 2004, Appl. En viron. Microbiol., 70.1088-1096). These results, although encouraging, at least in view of symptomatology, did not prove regression of histopa thological damage. Wei et al. (Wei, J. and Hemmings, G. P.; Medical Hypotheses, (2005), 64, 547-552) establish a genetic relationship between celiac disease and schizophrenia and report the beneficial effect of regression of schizophrenic symptoms in celiac patients treated with gluten-free diet (De Santis, A., et al.; J. Intern. Med. 1997; 242. 421-3). The use of certain lactic acid bacteria in the manufacture of baked goods is already known. US 4,140,800, to Kline, discloses a process for making a freeze-dried sourdough bakery starter composition, which uses Lactobacillus san francisco, with the aim to provide a product useful in the preparation of French bread. Desirably, the flour is high gluten. The solution provided by Di Cagno et al., of using sourdough lactic acid bacteria still leave some practical problems unresolved. In particular, (i) selected strains are the results of a very long time consuming re search which showed marked differences at strain level within the above species of sourdough lactic acid bacteria; (ii) the same results are obviously non reproducible at bakery plant; and, especially (iii) the se lected strains are not commercially available. Other references disclose the use of lactic acid bacteria and Bifidobac teria in food manufacturing. EP 0 856 259 relates to a composition for feed use containing a mixture of lyophilized live bacteria comprising at least two species of bacteria selected from Bifidobacteria and at least two species of bacteria se lected from Lactobacillus acidophilus, Streptococcus thermophilus, WO 2006/097415 PCT/EP2006/060505 4 Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus plantarum and Streptococcus faecium and one or more oligosaccharides. The com position is added to a liquid, creamy or pasty foodstuff, said foodstuff being a milk, a milk-based or milk-derived product, or a product based on or derived from vegetable products, said supplementation being carried out at the moment of use of the foodstuff. The product is not used in bakery. WO 03/071883 relates to dietetic and/or pharmaceutical compositions for human and/or animal use, and general foodstuffs, based on micro bial cultures consisting of autochthonous and allochthonous species with respect to human beings and animals, selected from species of lactic bacteria, propionibacteria, yeasts and/or molds. They have an equilibrating action of the intestinal flora of the host human being or animal, as well as having various beneficial/probiotic effects towards the host organism. There is no indication of a possible use in celiac dis ease. US 2004/265291 provides compositions, kits, and methods for provid ing or restoring beneficial bacteria to a subject. The compositions and kits optionally include food or nutrients to promote growth and prolif eration of the bacteria in the subject or an antimicrobial agent to re duce the presence of undesirable or pathogenic microbes in the subject. WO 02/065842 relates to starter preparations suitable for all types of cereal and the use of the same for producing bread and bakery prod ucts based on leaven or using leaven, especially for producing gluten free bakery products for people with coeliac disease. There is no dis closure of particular mixtures of lactic acid bacteria and Bifidobacteria. US 5,185,165 discloses a precursor base for use in a bakery dough product comprising an acidic concentrate, at least one type of sugar, yeast, at least one type of flour, non-fat dry milk and at least one type of lactic acid producing bacteria and a process for producing the pre cursor base are disclosed. The precursor base is useful in a process for WO 2006/097415 PCT/EP2006/060505 5 producing a precursor slurry (or active ferment concentrate) for use in making a preferment dough mixture for the preparation of the bakery dough product. In addition, processes for preparing the precursor slurry and the preferment dough mixture and an apparatus for produc ing the preferment dough mixture are disclosed. The reference to lac tica acid bacteria is totally generic. US 2004/110270 describes a bacterial composition having immuno modulation properties comprising at least one strain selected from the group consisting of Lactobacillus acidophilus PTA-4797, Lactobacillus plantarum PTA-4799, Lactobacillus salivarius PTA-4800, Lactobacil lus paracasei PTA-4798, Bifidobacterium bifidum PTA-4801 and Bifi dobacterium lactis PTA-4802. EP 1 258 526 discloses the production of a starter for making wheat predough and wheat sourdough by partially fermenting a mixture of water and milled wheat product(s) with an inoculum comprising lacto bacilli and yeast comprises using an inoculum comprising an adapted mixed flora including at least one yeast strain, at least one homofer mentative lactobacillus strain and at least one heterofermentative lac tobacillus strain. There are provided strains of Saccharomyces sp. DSM 14265, Lactobacillus pontis DSM 14269, Lactobacillus pontis DSM 14272, Lactobacillus pontis DSM 14273, Lactobacillus pontis DSM 14274, Lactobacillus crispatus DSM 14271, Lactobacillus planta rum DSM 14268 and Lactobacillus sanfranciscensis DSM 14270; an adapted mixed flora comprising Saccharomyces sp. DSM 14265 and at least three of Lactobacillus pontis DSM 14269, Lactobacillus pontis DSM 14272, Lactobacillus pontis DSM 14273, Lactobacillus pontis DSM 14274, Lactobacillus crispatus DSM 14271, Lactobacillus planta rum DSM 14268 and Lactobacillus sanfranciscensis DSM 14270. This reference pertains to the general technical filed of bakery products, with no special medical indications. WO 99/09839 relates to a paste-like composition which is applicable for use as such and as a filling, coating or other component of various food WO 2006/097415 PCT/EP2006/060505 6 products, and which contains a significant amount of probiotic. The food product is preferably a bakery product, in particular a rye containing bread, rusk, biscuit or the like. This reference deals with the generally known aspects of use of probiotics. The need of a baked product suitable for subjects affected by celiac dis ease, which can be obtained with an easy, reproducible process of manufacture and with materials reliable, safe and commercially avail able is still felt in this field. The present invention meets these needs by providing a baked product for subjects affected by celiac disease. However, the baked product finds a general usefulness in human diet because of its higher digesti bility. Summary of the invention It has now been found that certain specific mixtures of lactic acid bac teria and Bifidobacteria, of human and milk origin are endowed with the surprising property of being capable of hydrolyzing gliadin and glutenin fractions which are responsible for celiac disease. These specific mixtures are very useful in the manufacturing of sour dough and provide well-defined bacterial species. Therefore, it is an object of the present invention the use of specific mixtures of lactic acid bacteria and Bifidobacteria for the manufacture of sourdough. Another object of the present invention is represented by cereal-based food, in particular baked goods which are generally more digestible and in particular can be tolerated by CS patients. Another object of the present invention is a method for manufacturing cereal-based food, in particular baked goods suitable for subjects af- WO 2006/097415 PCT/EP2006/060505 7 fected by celiac disease and suitable to prevent contamination of glu ten in gluten-free products. A further object of the present invention is represented by gluten-free cereal-based food, in particular baked goods made of wheat flour when the use of the specific mixtures of lactic acid bacteria and bifidobacte ria is implemented under specific conditions with microbial proteolytic enzymes, routinely used in the bakery industries Another object of the present invention is a food for subjects affected by celiac disease, said food containing the specific mixture of lactic acid bacteria and Bifidobacteria herein disclosed. Still another object of the present invention is the use of the above mentioned mixtures of lactic acid bacteria and Bifidobacteria for the preparation of a product useful for reducing Platelet Activating Factor (PAF) and other inflammatory cytokines. These and other objects of the present invention will be now disclosed in detail in the foregoing, also by means of examples and Figures, wherein: Figure 1 shows 2DE analysis of gliadin protein fractions of different doughs made of wheat flour. (A) Chemically acidified dough (control). Prolamin polypeptides were indicated by numbered red ovals. (B) Dough incubated for 24 h at 37*C with MIXTURE 1 of the Example be low. Prolamin polypeptides were indicated by numbered red ovals. Blue numbers refer to polypeptides which were degraded more than 80%. Mr, molecular mass. Figure 2 shows hydrolysis of 33-mer peptide by MIXTURE 1 (10' cfu/ml). RP-FPLC at UV 214 nm trace of 200 gM 33-mer after 24 h of incubation at 37*C without microbial inoculum (A) and after 24 h of hydrolysis by MIXTURE 1 at 37*C (B).

WO 2006/097415 PCT/EP2006/060505 8 Detailed description of the invention According to the present invention, a mixture of at least 6, preferably at least 7, more preferably at least 8 species of lactic acid bacteria and/or Bifidobacteria selected from the group consisting of Lactobacil lus acidophilus, Lactobacillus buchneri, Lactobacillus casei, Lactoba cillus catenaforme, Lactobacillus cellobiosus, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus del brueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. lactis, Lac tobacillus helveticus, Lactobacillus jensenii, Lactobacillus leichmannii, Lactobacillus minutus, Lactobacillus paracasei, Lactobacillus planta rum, Lactobacillus rogosae, Lactobacillus salivarius, Lactobacillus brevis, Lactobacillus gasseri, Lactobacillus fermentum, Bifidobacte rium adolescentis, Bifidobacterium angulatum, Bifidobacterium bifi dum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobac terium dentium, Bifidobacterium eriksonii, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium plan tarum, Bifidobacterium pseudo-catenulatum, Bifidobacterium pseudo longum, Streptococcus lactis, Streptococcus raffinolactis, Acidamino coccus fermenta, Cytophaga fermentans, Rhodoferax fermentans, Cellu lomonas fermentans, Zymomonas mobilis, Streptococcus thermophilus are suitable for carrying out the present invention. Other species can be used, for example those disclosed in the state of the art and generally available in collections, such as ECACC, ASTM; DSM. The preferred mixtures according to the present invention are the fol lowing: Streptococcus thermophilus, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium breve, Lactobacillus acidophilus, WO 2006/097415 PCT/EP2006/060505 9 Lactobacillus plantarum Lactobacillus casei, Lactobacillus delbrueckii subsp. bulgaricus. Streptococcus thermophilus, Bifidobacterium lactis, Bifidobacterium breve, Lactobacillus acidophilus Lactobacillus plantarum, Lactobacillus casei, Lactobacillus helveticus. These mixtures of well known species can be easily prepared by any person having ordinary experience in this field. Conveniently, these mixtures are commercially available in a lyophi lized form. These formulations are suitable for use as starter in the preparation of sourdough. The cereal-based food, in particular baked goods obtained according to the present invention are generally more digestible, therefore are more accepted by the general consumer or particularly by people wishing or needing more digestible food. In a particular embodiment of the invention, the cereal-based food, in particular baked goods can be used for the integration of the diet of people affected by celiac disease, since gluten concentration is reduced to a low value and the amount of gluten which persisted in the dough is markedly hydrolyzed, especially for the peptide sequences which are responsible for CS. When one of the above microbial mixtures is integrated with a suffi cient amount of microbial protease, such as for example 200 ppm of WO 2006/097415 PCT/EP2006/060505 10 microbial protease (typically from Aspergillus sp.) under the conditions optimized in the present invention the fermented sourdough has a con centration of gluten lower than 200 ppm, as determined by using the monoclonal antibody R5. As stated by the Codex Alimentarius such type of product is defined gluten-free and therefore suitable for celiac patients. Microbial proteases are of common use in bakery, see for example WO 88/03365, EP 0588426, US 6,465,209, GB1,196,946. These proteases are commonly marketed, see for example Enzyme Development Corpo ration U.S.A. and the present invention can be carried out with any product available on the market and of common use in bakery. In a preferred embodiment of the present invention, the microbial pro tease is a fungal protease from Aspergillus oryzae; activity of 500,000 HUT/g; pH optimum about 3.0 and activity in the range of pH 3.0 to 6.0; temperature optimum about 50*C and activity in the range 25 60*C; or another protease is an acid stable protease from Aspergillus niger; activity of 3,000 SAPU/g; optimum pH 2.0-3.0 and activity in the range of pH 2,0 to 6,0; temperature optimum 50-60*C and activity in the range 30-60*C. These enzymes are available from Bio-Cat Inc., Troy, Virginia, U.S.A. and many other suppliers. The present invention allows making baked goods with a higher per centage of wheat flour, resulting in products with a more agreeable flavor and better accepted by people affected by celiac disease. The present invention also allows to obtain products directed to general consumers, including healthy people, endowed with better digestibil ity. In a wider aspect, the present invention also refers to starchy products comprising a mixture of lactic acid bacteria, optionally supplied with enzymatic preparations as disclosed above.

WO 2006/097415 PCT/EP2006/060505 11 In its widest aspect, the present invention provides a mixture of lactic acid bacteria and Bifidobacteria, optionally added with proteolytic en zymes of microbial origin, useful for the preparation of products for oral administration for improving digestion of gluten and gluten related substances. The sourdough comprising the specific mixture of the present inven tion is the critical aspect of the same. The sourdough is useful in a process for the preparation of a baked good, in particular bread, but this applies to all leavened and non leavened products, such as for example biscuits, pastries, cakes, pies, pizza, crackers, breadsticks, snacks and all other products known in the art. The sourdough according to the present invention is suitable also for preparations for making, also homemade making, cereal-based food, in particular baked goods. In this case, the package for a baked good will comprise, other than usual ingredients for the specific product, a leav ening preparation comprising the specific mixture of the present in vention. The leavening preparation according to the present invention can be a combination with the specific mixture of lactic acid bacteria and Bifi dobacteria or can be provided in the package separately with the lactic acid bacteria and Bifidobacteria mixture and will be mixed with this latter at the moment of use, for example in water to form a leavening suspension. The mixture of lactic acid bacteria can be packaged in a single container alone or in admixture with the proteolytic enzymes (protease) disclosed above. Starchy products are generally well-known in the field and are part of the common knowledge, also among consumers and homemade cook ing. In particular, the present invention is applied to cereal-based products.

WO 2006/097415 PCT/EP2006/060505 12 Examples of starchy products are all kinds of pasta, noodles, such as fried instant noodles and wet noodles, snack products, tortillas, corn chips, extruded cereals and shredded cereals. Methods for making pasta are well-known in the art and reference is made just for example to Pasta and Semolina Technology, Edited by R.C. Kill and K Turnbull, Blackwell Science, 2001 and patents owned by Barilla. Methods for making Asian starchy products are also well known and just exemplary reference is made to Asian Food, Science and Technology, Edited by Catharina Y W. Ang, KeShun Liu and Yao Wen Huang, Technomic Publishing Company, Inc., 1999 and US 20020160093 to Kao Corporation and WO 99/65331, to Societe de Pro duits Nestl6 S.A. Today, most pasta is manufactured by continuous, high capacity ex truders, which operate on the auger extrusion principle in which kneading and extrusion are performed in a single operation. The manufacture of pasta includes dry macaroni, noodle, and spaghetti production. Pasta products are produced by mixing milled wheat, water, eggs (for egg noodles or egg spaghetti), and sometimes optional ingredients. These ingredients are typically added to a continuous, high capacity auger extruder, which can be equipped with a variety of dies that de termine the shape of the pasta. The pasta is then dried and packaged for market. Pasta products contain milled wheat, water, and occasionally eggs and/or optional ingredients. Pasta manufacturers typically use milled durum wheat (semolina, durum granulars, and durum flour) in pasta production, although farina and flour from common wheat are occa sionally used. Most pasta manufacturers prefer semolina, which con sists of fine particles of uniform size and produces the highest quality pasta product. The water used in pasta production should be pure, free from offflavors, and suitable for drinking. Also, since pasta is produced WO 2006/097415 PCT/EP2006/060505 13 below pasteurization temperatures, water should be used of low bacte rial count. Eggs (fresh eggs, frozen eggs, dry eggs, egg yolks, or dried egg solids) are added to pasta to make egg noodles or egg spaghetti and to improve the nutritional quality and richness of the pasta. Small amounts of optional ingredients, such as salt, celery, garlic, and bay leafs, may also be added to pasta to enhance flavor. Disodium phos phate may be used to shorten cooking time. Other ingredients, such as gum gluten, glyceryl monostearate, and egg whites, may also be added. All optional ingredients should be clearly labeled on the package. Durum wheat is milled into semolina, durum granular, or durum flour using roll mills. Semolina milling is unique in that the objective is to prepare granular middlings with a minimum of flour production. After the wheat is milled, it is mixed with water, eggs, and any other op tional ingredients. In the mixing operation, water is added to the milled wheat in a mixing trough to produce dough with a moisture content of approximately 31 percent. Eggs and any optional ingredients may also be added. Most modern pasta presses are equipped with a vacuum chamber to remove air bubbles from the pasta before extruding. If the air is not removed prior to extruding, small bubbles will form in the pasta which diminish the mechanical strength and give the finished product a white, chalky appearance. After the dough is mixed, it is transferred to the extruder. The extru sion auger not only forces the dough through the die, but it also kneads the dough into a homogeneous mass, controls the rate of production, and influences the overall quality of the finished product. Although construction and dimension of extrusion augers vary by equipment manufacturers, most modern presses have sharpedged augers that have a uniform pitch over their entire length. The auger fits into a grooved extrusion barrel, which helps the dough move forward and re duces friction between the auger and the inside of the barrel. Extrusion barrels are equipped with a water cooling jacket to dissipate the heat WO 2006/097415 PCT/EP2006/060505 14 generated during the extrusion process. The cooling jacket also helps to maintain a constant extrusion temperature, which should be approxi mately 51*C (124*F). If the dough is too hot (above 74*C [165*F]), the pasta will be damaged. Uniform flow rate of the dough through the extruder is also important. Variances in the flow rate of the dough through the die cause the pasta to be extruded at different rates. Products of nonuniform size must be discarded or reprocessed, which adds to the unit cost of the product. The inside surface of the die also influences the product appearance. Until recently, most dies were made of bronze, which was relatively soft and required repair or periodic replacement. Recently, dies have been improved by fitting the extruding surface of the die with Teflon@ inserts to extend the life of the dies and improve the quality of the pasta. Drying is the most difficult and critical step to control in the pasta pro duction process. The objective of drying is to lower the moisture con tent of the pasta from approximately 31 percent to 12 to 13 percent so that the finished product will be hard, retain its shape, and store with out spoiling. Most pasta drying operations use a preliminary drier immediately af ter extrusion to prevent the pasta from sticking together. Predrying hardens the outside surface of the pasta while keeping the inside soft and plastic. A final drier is then used to remove most of the moisture from the product. Drying temperature and relative humidity increments are important factors in drying. Since the outside surface of the pasta dries more rap idly than the inside, moisture gradients develop across the surface to the interior of the pasta. If dried too quickly, the pasta will crack, giv ing the product a poor appearance and very low mechanical strength. Cracking can occur during the drying process or as long as several weeks after the product has left the drier. If the pasta is dried too WO 2006/097415 PCT/EP2006/060505 15 slowly, it tends to spoil or become moldy during the drying process. Therefore, it is essential that the drying cycle be tailored to meet the requirements of each type of product. If the drying cycle has been suc cessful, the pasta will be firm but also flexible enough so that it can bend to a considerable degree before breaking. Packaging keeps the product free from contamination, protects the pasta from damage during shipment and storage, and displays the product favorably. The principal packaging material for noodles is the cellophane bag, which provides moisture-proof protection for the prod uct and is used easily on automatic packaging machines, but is difficult to stack on grocery shelves. Many manufacturers utilize boxes instead of bags to package pasta because boxes are easy to stack, provide good protection for fragile pasta products, and offer the opportunity to print advertising that is easier to read than on bags. Air emissions may arise from a variety of sources in pasta manufactur ing. Particulate matter (PM) emissions result mainly from solids han dling and mixing. For pasta manufacturing, PM emissions occur dur ing the wheat milling process, as the raw ingredients are mixed, and possibly during packaging. Emission sources associated with wheat milling include grain receiving, precleaning/handling, cleaning house, milling, and bulk loading. Other information are available in D. E. Walsh and K. A. Gilles, "Pasta Technology", Elements Of Food Tech nology, N. W. Desrosier, Editor, AVI Publishing Company, Inc., 1977 The present invention is applicable both to the industrial manufacture and the home preparation of pasta, in the latter case, favourably in the preparation of egg pasta. According to the present invention, in the process of making dough, the mixture of lactic acid bacteria herein disclosed are used.

WO 2006/097415 PCT/EP2006/060505 16 In another embodiment of the present invention, typical Asian, cereal based food is provided. A preferred example is a kind of noodle known as Ramyun in Korea, Ramien in China and Ramen in Japan. As in the general carrying out of the present invention, the dough is prepared by adding the mixture of lactica acid bacteria and letting the sufficient time for pre-fermentation. The mixtures of lactic acid bacteria and Bifidobacteria according to the present invention, optionally added with the above mixtures of micro bial proteases, can also be used in the manufacture of a food, in par ticular gluten-free grade, for consumption by a subject affected by ce liac disease. Examples of this kind of food are pastas, cereals, tacos, tortillas, popcorn. For a reference see Practical Gastroenterology April 2004, pages 86-104 and the literature cited therein. Another object of the present invention is a method for the manufac ture of a baked good comprising the addition of the above sourdough preparation. In a preferred embodiment of the present invention, the method com prises the following steps: a) liquid pre-fermentation of 20-50% of wheat flour by weight (whole mixture 20%-50% of flour and 80%-50% of water, dough yield about 300 with about 109 cells of the mixture of the present invention per g of dough), at about 37 0 C for at least about 24 h, preferably be tween about 24 and about 31 hours; b) after fermentation, mixing the dough with one or more tolerated flour, such as millet flour, to have a final dough yield of about 150 (solid dough) and added of commercial baker's yeast at a concentration of about 1% by weight; c) incubating the dough at about 37 0 C for about 2 hours until the leavening is completed; d) baking at about 250 0 C for about 20 minutes.

WO 2006/097415 PCT/EP2006/060505 17 In a second embodiment of the present invention, the method can be modified as follows: a) liquid fermentation of 20% of wheat flour with the further addition of fungal proteases (200 ppm) at 37*C for 24-31 hours; b) after fermentation, drying to remove water in order to have a glu ten-free wheat flour (< 200 ppm of gluten); c) use of the wheat flour gluten-free as basic ingredient for the manu facture of cereal-based food, in particular baked goods. The term "about" in this circumstance means those values around those indicated which are comprised in normal carrying out of the in vention and can depend on the instrumental errors of the measuring devices or deviations made by the person skilled in the art around the indicated values, but that do not affect the result obtained by the in vention. The above ranges are intended also as about higher than the lower limit and about lower than the upper limit. Therefore, the liquid pre fermentation of step a) comprises an amount of wheat flour not lower than about 20% and not higher than 50% by weight for a time of not less than about 24 hours and not more than about 31 hours. An exemplary list of tolerated flours comprises bean flours, buckwheat, flax, corn (maize), legume flours (garbanzo/chickpea, lentil, pea), mil let, Indian Rice Grass, nut flours (almond, hazelnut, pecan), quinoa, potato flour, sweet potato flour, sago, seed flours (sesame), sorghum, soy, tapioca, teff. Millet is one of the preferred tolerated flours. A very advantageous embodiment of the present invention is to include a prebiotic in the baked good, whether containing or not a tolerated flour. A prebiotic is a non-digestible fibre-like substance, examples of which are short chain and long chain oligosaccharides, such as fructo- WO 2006/097415 PCT/EP2006/060505 18 oligosaccharides, soy-oligosaccharides, xylo-oligosaccharides and iso malto-oligosaccharides. An even more advantageous embodiment of the invention is the incor poration of the baked good herein disclosed in a baked good such as the one disclosed in EP 1 010 372. In this embodiment, the baked good comprises a non-baked, essentially water-free, fat-based composition comprising live lyophilized lactic bacteria. This fat-based composition comprising live lyophilized lactic bacteria can of course be combined with all the baked products of the present invention. The cereal-based food, in particular baked goods and the packages for the making cereal-based food, in particular baked goods according to the present invention are suitable for administration to a subject suf fering from celiac disease. As said above, the present invention comprises also general food known under the general name of starchy food, in particular cereal based food. The mixture of lactic acid bacteria, optionally added with the microbial protease, as previously described, is used in the manufacture of starchy food, in particular cereal-based food to obtain the same results and advantages of the above described embodiment of baked goods. So to say, the food obtained according to the present invention is suitable for subjects suffering from celiac disease or for general consumers, also in good health, wishing more digestible food. For example children and ageing people may wish more digestible food. Therefore, a further object of the present invention is a method for treating a subject suffering from celiac disease comprising the integra tion of the diet of said subject with a baked good and/or a starchy food as disclosed above. In the foregoing, the baked goods and the starchy food according to the present invention will be comprised in the term "cereal-based food".

WO 2006/097415 PCT/EP2006/060505 19 In another embodiment of the present invention, the cereal-based food, in particular baked goods can also be used for maintaining gluten tol erance or for inducing gluten tolerance or for decreasing the risk of al lergies due to wheat flour albumins and globulins. In another embodiment of the present invention, the cereal-based food, in particular baked goods can be safety used for celiac patients since the low concentration of gluten (< 200 ppm). The methods of treatment according to the present invention can also be used in combination with other medical treatments for celiac dis ease. As reported above, schizophrenic symptoms are noted in celiac patients and schizophrenic patients show sensitive behavior to gluten. The mix tures according to the present invention are useful for preparing glu ten-free dietetic goods. Therefore, a further object of the present invention is the use of the mixture disclosed above in the preparation of a gluten-free dietetic good useful for the treatment of schizophrenic symptoms. In particu lar, said symptoms affect a celiac or a non-celiac patient. Another problem in the art is the use of proline in preparations for en teric diet. In certain subjects, proline is not hydrolyzed and the com pounds making the solution for enteric diet are not assimilated. Also allergic responses can occur due to proline. The mixtures of lactic acid bacteria and Bifidobacteria disclosed in the present invention are use ful for hydrolyzing proline or proline-enriched peptides, thus making preparations for enteric diet effective and non-allergenic. Thanks to their properties, the mixtures of lactic acid bacteria and Bi fidobacteria disclosed in the present invention are also useful for mak ing gliadin-enriched glutamine solutions hypoallergenic.

WO 2006/097415 PCT/EP2006/060505 20 In another embodiment of the present invention, it has also been found that the mixtures herein disclosed can be used in the manufacture of a product for lowering the levels of Platelet Activating Factor (PAF) and other inflammatory cytokines for treating gastro-intestinal disease. PAF is involved in a series of gastro-intestinal diseases, in particular inflammatory disorders. We can mention ischemic bowel necrosis (Hsueh W, Gonzalez-Crussi F.; Methods Achiev. Exp. Pathol.; 1988.13; 208-39), gastric ulcer (Esplugues JV., Whittle B.J., Methods Find.; 1989. Supply. 1, 61-6), hemorrhagic rectocolitis (Chaussade S., Denizot Y, Ann. Gastroenterol. Hepatol. (Paris); 1991, May 27(3): 117-21), ne crotizing enterocolitis (Ewer AK, Acta Pediatr. Supply ; 2002, 91(437). 2-5; neonatal: Caplan MS., et al., Semin. Pediatr. Surg.; 2005, Aug 14(3): 154-51), inflammatory bowel disease (Nassif A., et al. Dis. Colon Rectum; 1996, Feb.; 39(2).217-23), pouchitis (Rothenberg DA., et al. Ann. Chir.; 1993; 47(10):1043-6). In view of the above explanation, the product according to the present invention can also be supplemented to subjects, in particular Japanese pople, having a deficit in PAF-hydrolase, who can be affected by a se ries of inflammatory diseases (Karasawa K; et al.; Prog. Lipid. Res.; 2003 Mar., 42(2):93-114). The product can take the form of a food, as disclosed above, or a nutri tional supplement, a nutraceutical, a drug. Nutritional supplement and nutraceutical are well-known terms in the art (Arvanitoyannis IS, et al.; Crit. Rev. Food Sci. Nutr.; 2005,45(5).385-404 and Kalra EK, AAPS PharmSci.; 2003, 5(3); E25) and there is no need of further definition. The following example further illustrates the invention. Example 1 Sourdough fermentation and electrophoresis analyses WO 2006/097415 PCT/EP2006/060505 21 The characteristics of the wheat flour used were as follows: moisture, 12.8%; protein (N x 5.70), 10.7% of dry matter (d.m.); fat, 1.8% of d.m.; ash, 0.6% of d.m.; and total soluble carbohydrates, 1.5% of d.m. Eighty grams of wheat flour and 190 ml of tap water (containing a cell concen tration of the cell preparations of about 109 cfu per g of dough) were used to produce 270 g of dough (dough yield, 220). Four doughs were manufactured by using the following mixtures of lactic acid bacteria and Bifidobacteria. Mixture 1 according to the invention: Streptococcus thermophilus, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium breve, Lactobacillus acidophilus, Lactobacillus plantarum Lactobacillus casei, Lactobacillus delbrueckii subsp. bulgaricus. Mixture 2 according to the invention Streptococcus thermophilus, Bifidobacterium lactis, Bifidobacterium breve, Lactobacillus acidophilus Lactobacillus plantarum, Lactobacillus casei, Lactobacillus helveticus. Mixture 3 Lactobacillus acidophilus, Lactobacillus brevis, Streptococcus thermophilus, Bifidobacterium infantis.

WO 2006/097415 PCT/EP2006/060505 22 Mixture 4 Lactobacillus brevis, Lactobacillus salivarius spp. salicinius Lactobacillus plantarum Fermentation was carried out at 37*C for 24 h. A dough without bacte rial inoculum was chemically acidified to pH 4.0 with a mixture of lac tic and acetic acids (molar ratio 4:1) and used as control. After incuba tion, gliadins were extracted from doughs following the method origi nally described by Osborne (Osborne, T.B.; 1970, The proteins of the wheat kernel. Carnegie Institute of Washington publication 84. Judd and Detweiler, Washington, D.C.) and further modified by Weiss et al. (Weiss, et al.; 1993, Electrophoresis, 14.805-816). Aliquots of 10-20 tl (about 10 tg of gliadin) were diluted 1:1 with sam ple buffer, treated at 100*C for 5 min and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to the Laemmli procedure (Laemmli; 1970, Nature, 227.680-685); the gels contained 12% of acrylamide and were stained with B10 Bio-Safe Coomassie blue (Bio-Rad Laboratories, Hercules, CA). Two dimensional gel electrophoresis (2DE) was performed as described by Di Cagno et al. (Di Cagno; et al., 2004). Three gels were analyzed, and spot intensities of chemically acidified dough (siCAD) and sourdough (added of MIXTURE 1) (siSD) were normalized as reported by Bini et al. (Bini, et al.; 1997, Electrophoresis, 18.2832-2841). The hydrolysis factor for individual proteins was expressed as [(siCAD - siSD)/siCAD] x 100. All the hydrolysis factors were calculated based on the average of the spot intensities of the three gels, and standard deviation was calculated. Only hydrolysis factors with statistical significance where P value was < 0.05 were reported. Hydrolysis of synthetic substrates, Pro-rich polypeptides and RP-FPLC analyses Preliminarily, the proline specific peptidase activities of Mixture 1 were characterized by using synthetic substrates such as Pro-p-NA, WO 2006/097415 PCT/EP2006/060505 23 Leu-p-NA, Ala-p-NA, Leu-Leu, Val-Leu, Pro-Gly, Gly-Pro-Ala, Leu Leu-Leu, Z-Gly-Pro-p-NA and NCBZ-Gly-Gly-Leu-p-NA (Sigma Chemical Co, St. Louis, Mo). The assay mixture contained 500 l of 200 mM phosphate buffer, pH 7.5, 150 l of substrate (0.2-3 mM, final concentration), 8 l of NaNa (0.05% final concentration) and 50 l of MIXTURE 1 preparation (5 x 109 cfu/ml, final concentration). Frag ment 62-75 (P-Q-P-Q-L-P-Y-P-Q-P-Q-S-F-P) of the A-gliadin (Silano and De Vincenzi; 1999) and the epitope 33-mer (L-Q-L-Q-P-F-P-Q-P-Q L-P-Y-P-Q-P-Q-L-P-Y-P-Q-P-Q-L-P-YP-Q-P-Q-P-F) (Shan et al., 2002) were chemically synthesized by Neosystem Laboratoire (Strasbourg, France). The assay mixture for the fragment 62-75 contained 320 l of 20 mM phosphate buffer, pH 7.0, 150 l of substrate (450 gM, final concentration), 8 l of NaNa (0.05% final concentration) and 50 l of MIXTURE 1 preparation (5 x 109 cfu/ml, final concentration). The as say mixture for the epitope 33-mer contained 500 l of 200 mM phos phate buffer, pH 7.5, 150 l of substrate (200 gM, final concentration), 8 l of NaNa (0.05% final concentration) and 50 l of MIXTURE 1 preparation (5 x 109 cfu/ml, final concentration). Both the mixtures were incubated at 37*C under stirred conditions (150 rpm). The en zyme kinetics for the hydrolysis of the 33-mer was calculated by using a Lineweaver-Burk plot (Lineweaver and Burk; 1934, J. American Chem. Soc., 56.658-666). The enzyme reactions were stopped by addition of 0.05% (vol/vol) (final concentration) trifluoroacetic acid. Peptides were separated from the mixture by RP-FPLC using a Resource II RPC 3ml column and FPLC equipment with a UV detector operating at 214 nm (Amersham Bio scences, Upssala, Sweden). Elution was at flow rate of 1 ml/min with a gradient (5 to 100%) of acetonitrile in 0.05% trifluoroacetic acid. The concentration of CH 3 CN was increased linearly from 5 to 46% between 16 and 62 min and from 46% to 100% between 62 and 72 min. The same procedure was used to determine the oligopeptides contained in the 70% ethanol-soluble extracts of the fermented doughs.

WO 2006/097415 PCT/EP2006/060505 24 Use of fungal proteases in association with lactic acid bacteria and Bi fidobacteria To produce a gluten-free sourdough (< 200 ppm), MIXTURE 1 was used in association with 200 ppm of fungal proteases routinely used in bakery products. During fermentation, the complementary activity of the proteolytic activities from bacteria and fungal sources gave a marked decrease of the of especially gliadin and glutenin fractions. The ethanol-soluble extracts of the fermented sourdough showed a glu ten concentration lower than 200 ppm as determined by the use of the monoclonal antibody R5. Western blot analysis with R5 monoclonal antibody and RAPD PCR analysis Fermented dough (37 0 C for 24 h) with MIXTURE 1 (about 109 cfu per g of dough) was mixed with non protein ingredients and tolerated flour (e.g., millet) to produce Italian biscuits and subjected to baking at 250 0 C for 15 min. Italian biscuits manufactured without fermentation with MIXTURE 1 were used as control. Biscuits were analyzed by Western blot R5 monoclonal antibody and RAPD PCR at the Centro National de Biotecnologia, Gluten Unit, CNB (28049 Madrid Spain). R5 monoclonal antibody recognizes potential toxic celiac peptides: QQPFP and 33-mer. RAPD PCR was carried based on specific DNA sequences which are related to potential toxic peptides. Hydrolysis of wheat flour salt soluble proteins (albumins and globu lins) Albumins and globulins were extracted from wheat flour by the method of Weiss (1993). The assay mixture, containing 0.8 ml of albu mins/globulins (about 3 mg/ml) in 50 mM Tris-HC1, pH 7.0, 5 x 109 cfu/ml of MIXTURE 1 and NaNa 0.05%. Incubation was at 37 0 C for 24 h under stirred conditions. A control without microbial cells was in cluded in the test. After incubation, the supernatant was recovered by WO 2006/097415 PCT/EP2006/060505 25 centrifugation and used for electrophoresis. Proteins from water/salt soluble fraction (albumins and globulins) were analyzed by im munoblotting (Curioni, A., et al., 1999, Clin. Exp. Allergy, 29.407-413) to detect the IgE binding of pooled sera from atopic patients, previ ously characterized as suffering from gastrointestinal symptoms re lated to wheat ingestion. By using a semidry blotting, protein bands, separated by SDS-PAGE, were transferred onto nitrocellulose sheets with a Trans-blot Cell (Bio-Rad Laboratories, Milan, Italy) with a transfer buffer containing 48 mM Tris, pH 9.2, 39 mM glycine, 20% methanol and 0.1% SDS, for 5 h at the voltage of 50 V. Blotting bands were visualized by soaking the membranes for a few minutes in Pon ceau S (0.1% in 3% trichloroacetic acid) and marked with a pencil, be fore destaining with water. Membranes were blocked with TBS con taining 0.05% Tween 20 (TBS-T) and 5% skim milk powder (M-TBS-T) for 2 h, and incubated overnight with pooled sera from patients, di luted 1:20 in TBS-T. After washing five times with M-TBS-T, blots were incubated for 1 h with monoclonal antihuman IgE peroxidase conjugate antibody (Sigma Chemical Co), diluted 1:5000 in M-TBS-T (Curioni, et al.; 1999). After four washes in M-TBS-T and one in TBS, bound IgE were visualized by chemiluminescence using the Supersig nal Detection kit (Pierce Biotechnology Inc., Rockford, IL), according to the instructions provided by the manufacturer. The procedure was car ried out at room temperature. Compared to the control, the SDS-PAGE profiles of the gliadin frac tions extracted from the doughs fermented with the four cell prepara tions showed that not all the cell preparations had the same capacity to degrade gliadins. Hydrolysis was very high for MIXTURE 1 of the invention, just slight for MIXTURE 2, while the other cell preparations (MIXTURES 3 and 4) did not cause an appreciable degradation. The differences among the four cell preparations were confirmed by the RP-FPLC analysis of the 70% ethanol soluble protein fractions which gave an overall view of the oligopeptides with apparent molecu lar masses lower than those detectable by electrophoresis.

WO 2006/097415 PCT/EP2006/060505 26 The above results gave a great evidence of the highest performance of the MIXTURE 1 which seemed to have a proteolytic activity more spe cifically addressed to gliadins. When the bacterial species which composed MIXTURE 1 were used in dividually at the same concentration of about 109 cells per g of dough, none of the 8 species gave a marked hydrolysis as shown by the mix ture. This was the first evidence of the complementary proteolytic ac tivity between the species of at least 6 strains which are used in MIXTURE 1 in well defined proportion. Gliadins and related oligopeptides are characterized by a large propor tion of proline residues within their sequences (Wieser, 1996, Acta Pe diatr. Supply. 412.3-9). Proline is unique among the 20 amino acids be cause of its cyclic structure. This specific conformation imposes many restrictions on the structural aspects of peptides and proteins, making them extremely resistant to hydrolysis. To adequately deal with such peptides, a group of specific peptidases is necessary to hydrolyze all the peptide bonds in which a proline residue occurs as potential sub strate at the different positions (Cunningham and Connor; 1997, Bio chim. Biophys. Acta, 1343.160-186). Preliminarily, the proline specific peptidase activities of MIXTURE 1 were characterized by using syn thetic substrates such as Pro-p-NA, Leu-p-NA, Ala-p-NA, Leu-Leu, Val-Leu, Pro-Gly, Gly-Pro-Ala, Leu-Leu-Leu, Z-Gly-Pro-p-NA and NCBZ-Gly-Gly-Leu-p-NA which are relatively specific for proline imin opeptidase, aminopeptidase, dipeptidase, prolinase, prolidase, dipepti dyl peptidase, tripeptidase, prolyl-endopeptidase and endopeptidase enzymes, respectively (Table 1). Table 1 Enzyme activities of MIXTURE 1. Substrate Type of enzyme Substrate concentra- Unit of activity tion (mM)

(U)

WO 2006/097415 PCT/EP2006/060505 27 Pro-p-NA Proline iminopeptidase 2 3.2 ± 0.02 Leu-p-NA Aminopeptidase 2 8.4 ± 0.04 Ala-p-Na Aminopeptidase 2 12.3 ± 0.05 Leu-Leu Dipeptidase 2 15.51 ±0.03 Val-Leu Dipeptidase 2 17.22 ±0.07 Pro-Gly Prolinase 3 8.0 0.02 Val-Pro Prolidase 2 3.03 0.02 Gly-Pro-Ala Dipeptidyl peptidase IV / 2 2.73 0.01 carboxypeptidase P Leu-Leu-Leu Tripeptidase 2 10.63 0.41 Z-Gly-Pro-p-NA Prolyl-endopeptidase 2 1.3 ± 0.01 NCBZ Gly-Gly- Endopeptidase 2 1.9 ± 0.02 Leu-p-NA Each value is the average of three enzyme assays, and standard deviations were calculated. A unit of enzyme activity (U) on p-NA substrates was defined as the amount of enzyme which produced an increase in absorbance at 410 of 0.01/min. A unit on polypeptides was the amount of enzyme which liberates 1 micromole of substrates/ min. All the above enzyme activities were largely distributed in the MIXTURE 1 preparation. Since it is very rare that a unique microbial strain may possess all the previous enzyme activities (Cunningham and O'Connor; 1997; Kunjii, et al.; 1996, Antoine Van Leeuwenhoek 70.187-221; Di Cagno et al.; 2004), only a pool of selected bacteria such as those contained in the MIXTURE 1 may have the complete pattern of peptidases needed for hydrolysis of Pro-rich oligopeptides. The hydrolysis of gliadin oligopeptides by MIXTURE 1 preparation during dough fermentation was further characterized by 2DE analysis. Eighty-four protein spots were identified in the chemically acidified dough used as control (Figure 1A). Seventy-nine of the 84 gliadin oli gopeptide spots were markedly degraded after dough fermentation with MIXTURE 1 compared to control (Figure 1B). Table 2 refers to the hydrolysis factors of the spots identified by 2DE. Most of the oli gopetides degraded (65 of the 79) had hydrolysis factors higher than 80% and only 8 showed hydrolysis factors lower than 40 %. Table 2 WO 2006/097415 PCT/EP2006/060505 28 Properties of alcohol-soluble polypeptides hydrolyzed by MIXTURE 1 after dough incubation at 37 0 C for 24 ha. Spot designationb Estimated pI Estimated molecular mass (kDa) Hydrolysis factor 1 6.84 51.0 54.0 2 7.15 49.8 90.5 3 6.55 49.5 85.0 4 6.38 49.0 92.0 5 7.52 48.9 95.4 6 9.40 48.7 87.0 7 7.64 48.5 90.6 8 7.98 48.4 85.0 9 8.05 48.3 90.8 10 9.52 48.1 96.2 11 9.15 48.0 91.5 12 9.87 47.9 90.0 13 9.70 47.8 97.7 14 6.83 47.7 85.0 15 9.10 47.6 92.5 16 9.69 47.0 90.8 17 9.25 46.3 90.8 18 7.08 46.0 52.5 19 8.70 44.5 93.2 20 6.54 44.0 95.6 21 6.63 43.2 0.0 22 7.10 43.0 10.0 23 6.70 42.9 91.4 24 8.04 42.6 67.0 25 6.35 42.5 0.0 26 6.04 41.8 0.0 27 6.49 41.7 87.7 28 6.40 41.6 16.0 29 6.78 41.4 95.0 30 7.00 41.3 47.5 31 7.58 41.2 93.2 32 8.55 41.1 90.1 33 8.45 41.0 85.4 34 8.25 40.9 86.2 35 8.00 40.8 20.5 36 8.68 40.7 93.1 37 8.85 40.65 88.6 38 8.90 40.6 84.8 39 9.18 40.55 81.5 40 6.37 40.5 45.6 41 6.56 40.4 82.0 42 7.20 40.0 93.0 43 6.05 39.9 95.2 44 6.26 39.8 24.8 45 6.48 39.7 95.0 46 6.57 39.6 44.5 47 6.81 39.5 93.5 48 9.55 39.3 91.7 WO 2006/097415 PCT/EP2006/060505 29 49 7.57 39.0 92.4 50 7.95 38.9 87.9 51 7.80 38.7 92.0 52 8.05 38.6 58.2 53 9.20 38.5 90.8 54 6.62 38.3 0.0 55 6.26 38.2 90.7 56 7.12 38.1 94.8 57 9.64 38.0 93.1 58 8.08 37.9 94.5 59 6.60 37.8 85.7 60 6.26 37.5 91.5 61 6.40 37.3 90.4 62 7.10 37.1 94.8 63 8.06 36.7 91.2 64 9.20 36.5 90.0 65 6.61 36.3 95.7 66 6.85 36.0 90.0 67 8.75 35.8 95.2 68 6.15 35.7 24.8 69 9.65 35.6 95.0 70 9.00 35.5 44.5 71 8.20 35.2 93.5 72 8.48 34.9 91.7 73 8.60 34.7 95.0 74 8.98 34.5 87.9 75 9.14 34.3 82.0 76 9.37 34.1 85.0 77 9.60 33.9 90.8 78 9.51 33.6 0.0 79 8.90 33.0 90.7 80 7.15 32.2 94.8 81 9.45 30.3 90.5 82 9.46 29.3 88.5 83 9.47 28.0 94.7 84 9.48 26.6 96.5 aAnalyses were performed with Image Master software (Pharmacia). Four gels of independent replicates were analyzed. For spot quantification and hydrolysis factor calculation, see Materi als and Methods. All of the hydrolysis factors were calculated based on the average of the spot intensities of each of four gels, and standard deviations were calculated. bSpot designation cor responds to those of the gels in Figure 1A and 1B. The above results showed that MIXTURE 1 had the capacity to almost totally hydrolyzed gliadin oligopeptides. The activity of MIXTURE 1 was further in vitro characterized towards some of the oligopeptides reported in the literature as the major re sponsible for CS: the fragment 62-75 of the A-gliadin (Silano and De Vincenzi; 1999) and the epitope 33-mer (Shan, et al.; 2002). As shown WO 2006/097415 PCT/EP2006/060505 30 by the RP-FPLC analysis, the fragment 62-75 of the A-gliadin, at a concentration of 450 gM, was completely hydrolyzed after 6 h of incu bation with 5 x 109 cfu/ml cells of MIXTURE 1. The epitope 33-mer, at a concentration of 200 gM, was completely hydrolyzed after 24 h of in cubation with the same cell concentration of MIXTURE 1 (Figure 2). The kinetics of hydrolysis of the 33-mer was determined by the Lineweaver-Burk plot showing a Vmax of 0.26 gmol per milliliter per min and a Km of 216 gM. As previously reported in the literature, it should be noted that the epitope 33-mer has the following properties: (i) it remains intact despite prolonged exposure to gastric and pancre atic proteases; (ii) it shows a hydrolysis less than 20% over 20 h of in cubation with small brush border membrane enzymes; and (iii) it re mains intact for a long time (about 24 h) in the small intestine and even at low concentration acts as potential antigen for T-cell prolifera tion (Shan, et al. 2002). The above results showed that MIXTURE 1 contained the complex pool of enzyme activities needed to completely hydrolyze the 33-mer and that these activities are markedly higher than those located at the gastrointestinal level. Compared to European gliadin references, the Western blot by R5 monoclonal antibody of Italian biscuits had the typical profile of intact gliadin. A major advantage of the R5 monoclonal antibody is its ability to recognize the consensus amino acid sequence QXPW/FP (Osman, et al.; 2001, Eur. J. Gastroenterol. Hepatol., 13. 1189-1193) corresponding to multiple immunoreactive epitope repeats, which occur in aX-, y- and o-gliadins as well as in different wheat varieties (Shewry, et al.; 1992, Cereal's proteins and celiac disease. In: Celiac disease, Marsh M. [ed], Oxford, Blackwell Scientific Publications pp.305-348). Greatest reactiv ity has been associated with the QQPFP amino acid sequence, but ho mologous repeats such as LQPFP, QLPYP, QLPTF, QQSFP, QQTFP, PQPPP, QQPYP and PQPFP are also recognized with a weaker reac tivity to R5 antibody (Osman, et al.; 2001). It is interesting to note that three of these epitopes (LQPFP, QLPYP and PQPFP) are placed in the sequence of the potent inducer of gut-derived human T-cell lines of ce liac patients, of the A-gliadin 33-mer peptide (Shan, et al.; 2002). The WO 2006/097415 PCT/EP2006/060505 31 Western blot of the Italian biscuits fermented with the MIXTURE 1 showed an almost degradation of ax-, P- and y-gliadins recognized by R5 monoclonal antibody. The same results were confirmed by RAPD PCR analysis. Preliminary experiments for the identification of allergen fractions of wheat albumins and globulins indicated that 100% of the sera tested were positive against albumin and globulin fractions. Responses were found against protein components with apparent molecular masses which ranged from 15 to 70 KDa, with an intense staining for some sera around 15 to 45 KDa. As determined by mono-dimensional SDS PAGE, the comparison of untreated albumins and globulins with that hydrolyzed by MIXTURE 1 preparation highlighted the hydrolysis of several potential allergens polypeptides. Example 2 The sourdough prepared according to Example 1 was used in the manufacture of baked products. Baked products as disclosed in Examples of US patent 6,884,443 were prepared by using the dough composition according to the present in vention instead of the one of the patent. Fermentation was carried out at 37*C for 24 hours as disclosed in Example 1 above and MIXTURE 1 was used. The products resulted more digestible and suitable for subjects af fected by celiac disease. Example 3 The sourdough prepared according to Example 1 with MIXTURE 2 was used in the manufacture of pasta.

WO 2006/097415 PCT/EP2006/060505 32 The pasta resulted more digestible and can be taken by subjects af fected by celiac disease. Example 4 MIXTURE 1 according to Example 1 was used in the manufacture of noodles according to the teaching of US 2002/0160093. Examples 1-4 of US 2002/0160093 were repeated, except a packet con taining a MIXTURE 1 according to the present invention was added to kansui and flour mixture. After kneading, the mixture was allowed to stand at 37*C for 24 hours. Then noodles were prepared as disclosed in the reference. The product resulted more digestible and suitable for subjects affected by celiac disease. Example 5 MIXTURE 2 according to Example 1 was used in the manufacture of noodles according to the teaching of WO 99/65331. Examples 1-2 of WO 99/65331 were repeated, except a packet contain ing a MIXTURE 2 according to the present invention was added to in gredient for the dough. After mixing, the dough was allowed to stand at 37*C for 24 hours. Then noodles were prepared as disclosed in the reference. The product resulted more digestible and suitable for subjects affected by celiac disease. Example 6 MIXTURE 1 according to Example 1 was used in the manufacture of bread derivatives according to the teaching of EP 0 614 609.

WO 2006/097415 PCT/EP2006/060505 33 Examples 1-5 of EP 0 614 609 were repeated, except a packet contain ing a MIXTURE 1 according to the present invention was added to dough preparation. After kneading, dough was allowed to stand at 37 0 C for 24 hours. Then products were prepared as disclosed in the ref erence. The product resulted more digestible and suitable for subjects affected by celiac disease. Example 7 MIXTURE 2 according to Example 1 was used in the manufacture of spaghetti according to the teaching of EP 1 338 209. Example of EP 1 338 209 was repeated, except a packet containing a MIXTURE 2 according to the present invention was added to ingredi ent for the dough. After mixing, the dough was allowed to stand at 37 0 C for 24 hours. Then noodles were prepared as disclosed in the ref erence. The product resulted more digestible and suitable for subjects affected by celiac disease. Example 8 Ramyun Composition: 1. Noodle Flour: 83-85% Refined Oil: 15-18% Salt : 1% Others: 0.6-1% WO 2006/097415 PCT/EP2006/060505 34 2. Dry Soup Base Dried Beef Flake, Soy Sauce, Mono Sodium Glutamate, Disodium Glu tamate, Flavoring Additive, Glucose, Garlic, Onion, Green Onion, Red Pepper Powder, Other ingredient for flavor Manufacturing Process for Ramvun Flour ( sometimes starch, rice flour, barley flour can be used in a dif ferent ratio) and water are mixed according to the manufacturer's rec ommendation. MIXTURE 1 was added to the dough and left to stand at 37*C for 24 hours. Roll the mixed dough with pressing roller and then put the dough through the machine to make the individual string of noodles. The shape and thickness of the noodle can be modified to the wanted thick ness and shape by adjusting the slot size of the shredding machine and also the speed of the convey belt carrying the noodle. Noodle pass through the steam box of which temperature is more than 100 * C to induce the pregelatinized starch ( - starch) to make the di gestion easier. After the steaming process, noodle is formed into the certain shape by molding case. Deep frying process: Depending on the type of Ramyun, the dehydra tion occurs through deep frying process. The noodle goes through deep frying process at 150*C. Some Ramyun does not go through this deep frying process. After the deep frying process, the Ramyun goes through cooling proc ess. According to the present invention, the specific mixture of lactic acid bacteria and Bifidobacteria is suitable for the manufacturing of cereal- WO 2006/097415 PCT/EP2006/060505 35 based food, in particular baked goods, which may be more tolerated by CS patients. In particular, the following advantages are provided: (i) marked capacity to degrade gliadin oligopeptides during dough fer mentation; (ii) hydrolysis of 79 of the 84 gliadin oligopeptides identified by 2DE analysis; (iii) complementary and large enzyme activities towards synthetic pep tides which included proline at the different positions; (iv) capacity to hydrolysis completely oligopeptides (fragment 62-75 of the A-gliadin and epitope 33-mer) which are responsible for CS; (v) capacity to markedly decrease ax-, P- and y-gliadins which reacted with R5 monoclonal antibody; (vi) capacity to hydrolyze several allergen polypeptides. (vii) when the activity of the above bacteria is supplemented with fun gal proteases and used towards 20% wheat flour under liquid fermen tation, it increases strongly producing gluten-free wheat flour.

Claims (46)

1. A sourdough comprising a mixture of at least 6 species of lactic acid bacteria and/or Bifidobacteria selected from the group consisting of Lactobacillus acidophilus, Lactobacillus buchneri, Lactobacillus ca sei, Lactobacillus catenaforme, Lactobacillus cellobiosus, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus delbrueckii, Lactoba cillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus helveticus, Lactobacillus jensenii, Lactobacillus leichmannii, Lactobacillus minutus, Lactobacillus paracasei, Lactoba cillus plantarum, Lactobacillus rogosae, Lactobacillus salivarius, Lac tobacillus brevis, Lactobacillus gasseri, Lactobacillus fermentum, Bifi dobacterium adolescentis, Bifidobacterium angulatum, Bifidobacte rium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium dentium, Bifidobacterium eriksonii, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium plantarum, Bifido bacterium pseudo-catenulatum, Bifidobacterium pseudolongum, Strep tococcus lactis, Streptococcus raffinolactis, Acidaminococcus fermenta, Cytophaga fermentans, Rhodoferax fermentans, Cellulomonas fermen tans, Zymomonas mobilis, Streptococcus thermophilus.

2. The sourdough according to claim 1, wherein said mixture of lac tic acid bacteria and Bifidobacteria comprises Streptococcus thermophi lus, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacte rium breve, Lactobacillus acidophilus, Lactobacillus plantarum, Lac tobacillus casei, Lactobacillus delbrueckii subsp. bulgaricus, or said mixture comprises Streptococcus thermophilus, Bifidobacterium lactis, Bifidobacterium breve, Lactobacillus acidophilus, Lactobacillus plan tarum, Lactobacillus casei, Lactobacillus helveticus.

3. The sourdough according to claim 1 or 2, further comprising at least one microbial proteolytic enzyme of use in bakery industry.

4. The sourdough according to claim 3, wherein said proteolytic enzyme is of fungal or bacterial source. WO 2006/097415 PCT/EP2006/060505 37

5. The sourdough according to claim 4, wherein said proteolytic enzyme is obtained from Aspergillus sp.

6. A leavening composition comprising a mixture of at least 6 spe cies of lactic acid bacteria and/or Bifidobacteria selected from the group consisting of Bifidobacteria selected from the group consisting of Lactobacillus acidophilus, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus catenaforme, Lactobacillus cellobiosus, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus delbrueckii, Lactoba cillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus helveticus, Lactobacillus jensenii, Lactobacillus leichmannii, Lactobacillus minutus, Lactobacillus paracasei, Lactoba cillus plantarum, Lactobacillus rogosae, Lactobacillus salivarius, Lac tobacillus brevis, Lactobacillus gasseri, Lactobacillus fermentum, Bifi dobacterium adolescentis, Bifidobacterium angulatum, Bifidobacte rium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium dentium, Bifidobacterium eriksonii, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium plantarum, Bifido bacterium pseudo-catenulatum, Bifidobacterium pseudolongum, Strep tococcus lactis, Streptococcus raffinolactis, Acidaminococcus fermenta, Cytophaga fermentans, Rhodoferax fermentans, Cellulomonas fermen tans, Zymomonas mobilis, Streptococcus thermophilus.

7. The leavening composition according to claim 6, wherein said mixture of lactic acid bacteria and Bifidobacteria comprises Streptococ cus thermophilus, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium breve, Lactobacillus acidophilus, Lactobacillus plan tarum, Lactobacillus casei, Lactobacillus delbrueckii subsp. bulgaricus, or said mixture comprises Streptococcus thermophilus, Bifidobacterium lactis, Bifidobacterium breve, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus helveticus.

8. The leavening composition according to claim 6 or 7, further comprising at least one microbial proteolytic enzyme of use in bakery industry. WO 2006/097415 PCT/EP2006/060505 38

9. The leavening composition according to claim 8, wherein said proteolytic enzyme is of fungal or bacterial source.

10. The leavening composition according to claim 9, wherein said proteolytic enzyme is obtained from Aspergillus sp.

11. The use of the sourdough of any one of claims 1-5 or the leaven ing composition of any one of claims 6-10 in the manufacture of a baked good.

12. A starchy food, in particolar a cereal-based food, comprising a mixture of at least 6 species of lactic acid bacteria and/or Bifidobacte ria selected from the group consisting of Lactobacillus acidophilus, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus catenaforme, Lactobacillus cellobiosus, Lactobacillus crispatus, Lactobacillus curva tus, Lactobacillus delbrueckii, Lactobacillus delbrueckii subsp. bulga ricus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus helveticus, Lactobacillus jensenii, Lactobacillus leichmannii, Lactobacillus minu tus, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rogosae, Lactobacillus salivarius, Lactobacillus brevis, Lactobacillus gasseri, Lactobacillus fermentum, Bifidobacterium adolescentis, Bifi dobacterium angulatum, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium dentium, Bifido bacterium eriksonii, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium plantarum, Bifidobacterium pseudo-catenulatum, Bi fidobacterium pseudolongum, Streptococcus lactis, Streptococcus raffi nolactis, Acidaminococcus fermenta, Cytophaga fermentans, Rhod oferax fermentans, Cellulomonas fermentans, Zymomonas mobilis, Streptococcus thermophilus.

13. The food according to claim 12, wherein said mixture of lactic acid bacteria and Bifidobacteria comprises Streptococcus thermophilus, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium breve, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacil lus casei, Lactobacillus delbrueckii subsp. bulgaricus, or said mixture WO 2006/097415 PCT/EP2006/060505 39 comprises Streptococcus thermophilus, Bifidobacterium lactis, Bifido bacterium breve, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus helveticus.

14. The food according to claim 12 or 13, which is a baked good.

15. The food according to claim 14, selected from the group consist ing of biscuits, pastries, cakes, pies, pizza, crackers, breadsticks, snacks, pasta, noodles, tortillas, corn chips, extruded cereals and shredded cereals, ramyun.

16. The baked good according to any one of claim 14-15, further comprising at least one microbial proteolytic enzyme of use in bakery industry.

17. The baked good according to claim 16, wherein said proteolytic enzyme is of fungal or bacterial source.

18. The baked good according to claim 17, wherein said proteolytic enzyme is obtained from Aspergillus sp.

19. A package for a making a baked good comprising usual ingredi ents and a leavening composition according to any one of claims 6-10.

20. A process for the preparation of a baked good comprising the addition of the sourdough preparation of any one of claims 1-5 to usual ingredients.

21. The process according to claim 20, comprising the following steps: a) liquid pre-fermentation of about 20-50% of wheat flour by weight (whole mixture 20%-50% of flour and 80%-50% of water, dough yield about 300) with about 109 cells of the mixture as described in any one of claims 1-5 per g of dough, at 37 0 C for at least about 24 h, pref erably between about 24 and about 31 hours; WO 2006/097415 PCT/EP2006/060505 40 b) after fermentation, mixing the dough with one or more tolerated flour, such as millet flour, to have a final dough yield of 150 (solid dough) and added of commercial baker's yeast at a concentration of about 1% by weight; c) incubating the dough at about 37 0 C for about 2 hours un til the leavening is completed; d) baking at 250 0 C for about 20 minutes.

22. The process according to claim 21, wherein, in step b) the dough is mixed with tolerated flour.

23. The process according to claim 22, wherein, said tolerated flour is selected from the group consisting of bean flours, buckwheat, flax, corn (maize), legume flours, millet, Indian Rice Grass, nut flours, qui noa, potato flour, sweet potato flour, sago, seed flours, sorghum, soy, tapioca, teff.

24. The process according to any one of claims 20-23, further com prising the addition of fungal proteases.

25. The process according to claim 24, when dependent on any one of claims 19-21, wherein, in step a), said addition is carried out at 37 0 C and fermented for 24-31 hours and wheat flour is 20% by weight.

26. A baked good obtainable by the process any one of claims 20-25.

27. A baked good, in particular as disclosed in claim 26, containing one or more tolerated flour.

28. The baked good according to claim 27, wherein said tolerated flour is selected from the group consisting of bean flours, buckwheat, flax, corn (maize), legume flours, millet, Indian Rice Grass, nut flours, quinoa, potato flour, sweet potato flour, sago, seed flours, sorghum, soy, tapioca, teff. WO 2006/097415 PCT/EP2006/060505 41

29. The food according to any one of claims 12-18 or 26-28, further comprising a prebiotic.

30. The food according to any one of claims 12-18 or 26-29, combined with an essentially water-free, fat-based composition comprising live lyophilized lactic bacteria.

31. A gluten-free product, or a product with a reduced level of gluten according to any one of claims 12-18 or any of claims 26-30.

32. A product for enteric diet comprising a mixture disclosed in any one of claims 1-5.

33. A gliadin-enriched glutamine solution comprising a mixture dis closed in any one of claims 1-5.

34. The use of the sourdough of any one of claims 1-5 and/or the leavening composition of any one of claims 6-10 for the manufacture of a food, in particular a baked good for the integration of the diet of a subject suffering from celiac disease.

35. The use of the sourdough of any one of claims 1-5 and/or the leavening composition of any one of claims 6-10 for the manufacture of a food, in particular a baked good for maintaining gluten tolerance in a subject suffering from celiac disease.

36. The use of the sourdough of any one of claims 1-5 and/or the leavening composition of any one of claims 6-10 for the manufacture of a food, in particular a baked good for inducing gluten tolerance in a subject suffering from celiac disease.

37. The use of the sourdough of any one of claims 1-5 and/or the leavening composition of any one of claims 6-10 for the manufacture of a food, in particular a baked good for decreasing the risk of allergies due to wheat flour albumins and globulins. WO 2006/097415 PCT/EP2006/060505 42

38. The use of the mixture disclosed in any one of claims 1-5 in the preparation of a gluten-free dietetic good useful for the treatment of schizophrenic symptoms.

39. The use according to claim 38, wherein said symptoms affect a celiac patient.

40. The use according to claim 38, wherein said symptoms affect a non-celiac patient.

41. The use of the mixture disclosed in any one of claims 1-5 in the preparation of products for enteric diet.

42. The use of the mixture disclosed in any one of claims 1-5 for the preparation of a product for reducing Platelet Aggregating Factor (PAF) and/or inflammatory cytokines in gastro-intestinal diseases.

43. The use according to claim 42, wherein said disease is an in flammatory disease.

44. The use according to claim 43, wherein said disease is selected from the group consisting of ischemic bowel necrosis, gastric ulcer, hemorrhagic rectocolitis, necrotizing enterocolitis, neonatal necrotizing enterocolitis, inflammatory bowel disease and pouchitis.

45. The use of the mixture disclosed in any one of claims 1-5 in the preparation of gliadin-enriched glutamine solutions.

46. The use of the mixture disclosed in any one of claims 1-5 in the preparation of a product for oral administration for improving diges tion of gluten and gluten-related substances.